Human telomeres (the structures that cap the ends of chromosomes) are composed of many kilobases of the repetitive sequence TTAGGG that together with telomere binding proteins prevent the cell from recognizing the ends as DNA breaks needing repair. Telomeres shorten due to incomplete DNA lagging strand synthesis/processing, and cellular senescence occurs when some have shortened sufficiently to induce a DNA damage signal. Cancer cells escape the proliferative limits of replicative aging by one of two mechanisms. Most frequently they up-regulate the expression of telomerase, an enzyme capable of adding telomere repeats to the ends of the chromosomes and maintaining their length. Much less frequently tumors activate a recombination-based ALT (Alternative Lengthening of Telomeres) mechanism for maintaining the ends of their chromosomes. The detailed structure of telomeres is very difficult to study because of the lack of restriction enzyme sites in the repetitive TTAGGG sequence (which prevents standard molecular biological approaches) and the fact that each diploid cell contains 92 telomeres (23 chromosomes x 2 ends x two copies of each). We have developed techniques that now permit us to address a large number of fundamental issues in telomere biology. In Specific Aim 1, we will determine how rapidly following replication the mature structure of the 3'overhangs are established, what factors influence the timing or extent of this processing, when telomerase acts to elongate the G-strand, how much telomerase can add, how telomerase action and the C-strand fill-in are coordinated, and how special structures called t- loops are unfolded and reformed during replication. Aim 2 explores the relationships between the mechanism that prevents restriction enzymes from digesting the region adjacent to the telomere and that which normally blocks recombination from occurring within the highly repetitive telomeric sequences. Investigations will include how these mechanisms are changed in cells using the ALT mechanism, and if many different types of ALT pathways exist. The results of these studies will not only advance our understanding of basic telomere regulation, but will also identify new therapeutic targets for inhibiting telomere maintenance in cancer (steps in telomerase elongation or ALT recombination) or slowing telomere shortening in age-related diseases. PUBLIC HEALTH RELEVANCE: Telomeres cap the ends of all of our chromosomes and protect them from degradation. Telomere shortening causes replicative aging, and cancer cells are immortal because they acquire mechanisms to prevent this. Knowledge of the structure and processing of telomeres and the mechanisms by which they are maintained will provide targets for the development of new drugs to treat cancer and age-related diseases.